Since the discovery that the endothelium derived relaxing factor (EDRF) was the endogenous toxic gas nitric oxide (NO), an astonishing number of physiological functions have been attributed to NO. Despite the widely recognized importance of NO, little is known about the mechanism of regulation of the NO receptor, the soluble guanylyl cyclase (sGC). sGC is a heme containing heterodimer that catalyzes the formation of cGMP from the substrate GTP. Upon binding of NO, the sGC is activated several hundred fold. The sGC is a multi-domain signaling enzyme that contains the NO receptor-heme domain, a dimerization domain and the effector-catalytic domain. It is not known how the NO signal is propagated to the catalytic domain of sGC. We recently discovered that sGC is desensitized by S-nitrosylation, the addition of a NO moiety to the free thiol of a specific cysteine (Cys). There s now substantial evidence that thiol redox reactions are critical and dynamic regulators of sGC function and that thiol modifications of sGC have clinical relevance as they are associated with decreased vascular reactivity in hypertension. However, the mechanisms by which modification of reactive Cys modulate sGC activation, NO signal transduction to the catalytic domain, sGC basal activity, domain interactions and NO-heme affinity have yet to be explored. The proposed studies seek to understand the structural and molecular basis of mechanisms of regulation of the sGC and how disruption of these mechanisms contributes to development of cardiovascular pathologies. We will use purified enzymes, cellular system and animals models to conduct structure-function studies, molecular dynamics simulation, biochemical and kinetics analysis and applied physiology for the following aims: 1) define the role of thiol Cys in the molecular mechanism of activation of sGC; 2) investigate the modulation of sGC by the thiol-reducing protein thioredoxin and 3) establish how thiol-dependent dysfunction of sGC contributes to hypertension and cardiac hypertrophy. Understanding the mechanisms of regulation of sGC will be key to uncovering the molecular basis of some types of hypertension, atherosclerosis and erectile dysfunction, which affect more than 60 millions Americans.